RESEARCH ARTICLE elifesciences.org The transcription factor Pitx2 positions the embryonic axis and regulates twinning Angela Torlopp1†‡, Mohsin A F Khan1†§, Nidia M M Oliveira1, Ingrid Lekk1, Luz Mayela Soto-Jiménez1,2¶, Alona Sosinsky3#, Claudio D Stern1* 1Department of Cell and Developmental Biology, University College London, London, United Kingdom; 2Programa de Ciencias Genómicas, Universidad Nacional Autónoma de México, Morelos, Mexico; 3Institute of Structural and Molecular Biology, Birkbeck College, University of London, London, United Kingdom *For correspondence: c.stern@ ucl.ac.uk †These authors contributed Abstract Embryonic polarity of invertebrates, amphibians and fish is specified largely by equally to this work maternal determinants, which fixes cell fates early in development. In contrast, amniote embryos Present address: ‡Cardiovascular remain plastic and can form multiple individuals until gastrulation. How is their polarity determined? Research Institute, University of In the chick embryo, the earliest known factor is cVg1 (homologous to mammalian growth California, San Francisco, differentiation factor 1, GDF1), a transforming growth factor beta (TGFβ) signal expressed San Francisco, United States; posteriorly before gastrulation. A molecular screen to find upstream regulators of cVg1 in normal §Cardiology and Thorax Surgery, embryos and in embryos manipulated to form twins now uncovers the transcription factor Pitx2 as a Faculty of Medical Sciences, candidate. We show that Pitx2 is essential for axis formation, and that it acts as a direct regulator of University of Groningen, cVg1 expression by binding to enhancers within neighbouring genes. Pitx2, Vg1/GDF1 and Nodal ¶ Groningen, Netherlands; Stark are also key actors in left–right asymmetry, suggesting that the same ancient polarity determination Lab, Research Institute of mechanism has been co-opted to different functions during evolution. Molecular Pathology, Vienna, DOI: 10.7554/eLife.03743.001 Austria; #Clinical Genome Informatics Facility, Imperial Centre for Translational and Experimental Medicine, Imperial College, London, United Kingdom Introduction In most invertebrates and anamniote vertebrates (fishes and amphibians), embryonic polarity is first Competing interests: The authors declare that no established by localisation of maternal determinants in the cytoplasm and/or cortex of the fertilised competing interests exist. egg. This generates differences between the blastomeres that will form by cell division from the egg, and which will culminate in specifying the orientation of the embryonic axes (Wilson, 1898). Separation Funding: See page 21 of the first two blastomeres can lead to twinning: the formation of genetically identical, complete Received: 22 June 2014 individuals (Driesch, 1892). Separation of blastomeres after the four-cell stage, however, does not Accepted: 14 November 2014 generate twins; in most cases it interferes with development of even a single embryo owing to the Published: 12 December 2014 removal of important determinants that have by then segregated to different cells. This is known as Reviewing editor: Marianne E the mosaic mode of development. Among the vertebrates, amniotes (birds and many mammals, and Bronner, California Institute of possibly also reptiles) have a remarkably extended capacity to give rise to twins. Some species of the Technology, United States armadillo genus Dasypus generate quadruplets or octuplets from a single fertilisation event, as a result of two or more sequential ‘splitting’ events of the embryo at a stage when it is already highly multi- Copyright Torlopp et al. This cellular (Newman and Patterson, 1910; Loughry et al., 1998; Enders, 2002; Eakin and Behringer, article is distributed under the 2004). Conjoined (‘Siamese’) twins occur in mammals including humans (Chai and Crary, 1971; terms of the Creative Commons Attribution License, which Vanderzon et al., 1998; Kaufman, 2004) and are also seen in reptiles (Cunningham, 1937) and birds permits unrestricted use and (Ulshafer and Clavert, 1979); most of these are thought to arise from splitting of the embryo rela- redistribution provided that the tively late in development (Kaufman, 2004). Perhaps the most dramatic example is seen in the chick, original author and source are where cutting an embryo into fragments at the blastoderm stage (when the embryo contains as many credited. as 20,000–50,000 cells) can lead to each fragment generating a complete embryo; up to eight embryos Torlopp et al. eLife 2014;3:e03743. DOI: 10.7554/eLife.03743 1 of 24 Research article Developmental biology and stem cells eLife digest In warm-blooded animals, including chickens and humans, a single embryo can give rise to several separate individuals (identical twins). Some species of armadillos routinely give birth to quadruplets in this way—and in experiments, up to eight identical chick embryos can be produced by cutting one embryo into smaller pieces (a type of ‘experimental twinning’). This ability of a developing embryo to subdivide into separate individuals ends when the embryo starts to form its first midline structure, called the ‘primitive streak’. This is the first line of symmetry and defines where the head–tail axis will later develop. The steps that establish the axes of the embryo in birds and mammals, and the factors that prevent further splitting of the embryo to form twins after this point, are only just beginning to be understood. In chick embryos, the production of a protein called cVg1 is the first known step and precedes the development of a line of symmetry. A similar protein is produced in mammalian embryos and both proteins are members of an important family of signalling proteins. Now, Torlopp, Khan et al. have used a combination of techniques to search for other proteins that that control the production of the cVg1 protein. Genes that are active in the region of the embryo that will express cVg1 later in development were identified, both in normal embryos and during the process of experimental twinning. This search revealed Pitx2 as a protein that acts to switch on the expression of the gene that encodes cVg1. When the Pitx2 protein is removed, the embryonic axis forms from the opposite side. Next, Torlopp, Khan et al. searched the chicken genome to identify stretches of DNA around the cVg1 gene where proteins that regulate gene expression might bind. Six potential sites were found, including four to which Pitx2 can bind. Further experiments confirmed that two of these regulatory sequences encourage the expression of the cVg1 gene at its correct position in the embryo. Pitx2 and related proteins were known to be involved with the development of left–right symmetry later in development; the findings of Torlopp, Khan et al. reveal, unexpectedly, that these proteins are also involved in first establishing the position at which the midline of the embryo will arise. It remains unclear what prevents most embryos from forming twins. But Torlopp, Khan et al.'s findings could help to explain some strange observations, made long ago, about left–right asymmetry in identical twins. For example, they could help explain why one of the twins in an identical twin pair is more likely to be left-handed than an individual in the general population, and why the direction of whorls of hair on the back of the head is often mirrored between identical twins. DOI: 10.7554/eLife.03743.002 have been generated from a single blastoderm by experimental splitting, right up to the time of appearance of the primitive streak (Lutz, 1949; Spratt and Haas, 1960). The ability of higher verte- brate embryos to retain a regulative model of development until such a late stage strongly suggests that localisation of maternally inherited determinants is not an essential component of the mechanisms specifying embryo polarity (Stern and Downs, 2012). Moreover, since a single blastoderm can gen- erate multiple embryos, mechanisms must exist that suppress this ability in regions of the embryo that do not normally initiate axis formation (Bertocchini and Stern, 2002; Bertocchini et al., 2004). In chick embryos, the earliest symmetry breaking event known is the localised expression of cVg1, the chick orthologue of mammalian growth differentiation factor 1 (GDF1)—a member of the trans- forming growth factor beta (TGFβ) superfamily of secreted proteins—encoding a Nodal/Activin-type molecule that signals through Smad2/3 (Weeks and Melton, 1987; Thomsen and Melton, 1993; Kessler and Melton, 1995; Seleiro et al., 1996; Shah et al., 1997; Kessler, 2004; Birsoy et al., 2006; Chen et al., 2006; Andersson et al., 2007). Before primitive streak stages, cVg1 is expressed in the posterior marginal zone (PMZ), an extraembryonic region adjacent to where the primitive streak will form; misexpression of cVg1 in other (anterior or lateral) parts of the marginal zone is sufficient to induce a complete axis from adjacent embryonic cells (Seleiro et al., 1996; Shah et al., 1997; Skromne and Stern, 2001, 2002). The mechanisms that position cVg1 in the PMZ are unknown. Moreover, when a blastoderm is cut in half at right angles to the future primitive streak axis, cVg1 expression spontaneously initiates in the marginal zone adjacent to the cut edge, in either the right or left side at equal frequency, foreshadowing the appearance of the primitive streak a few hours later (Bertocchini et al., 2004). This observation shows that the mechanisms that position cVg1 are active in the Torlopp et al. eLife 2014;3:e03743.